Therapeutic compositions against streptococcal infections, transformed hosts, methods of immunization and genetically engineered products

ABSTRACT

Non-virulent bacteria are disclosed into which have been cloned heterologous nucleotide sequences encoded for the expression of Streptococcal M protein antigens, which are effective to elicit opsonic antibodies against Streptococcal infections. These bacteria are useful for vaccination against Streptococcus pyogenes bacteria.

This is a division of application Ser. No. 07/088,626, filed Aug. 24,1987.

This invention relates to immunization against Streptococcal infections,more particularly against group A streptococcal infections.

The invention has broad and important implications. As far as is knownby the inventor, there is provided for the first time a biologicalliving vehicle which carries a protective antigen of a virulent bacteriawhich antigen is effective to immunize against infections caused by thevirulent bacteria. It is contemplated that the embodiments heredisclosed are applicable to broader and more far reaching applications,methods and uses.

The search for a safe and effective vaccine against those strains ofgroup A streptococci that trigger rheumatic fever and rheumatic heartdisease has been ongoing for more than sixty years. Lancefield, "CurrentKnowledge of Type-Specific M Protein Antigens to Group A Streptococci,"J. Immunol, Vol. 89, pp. 307-313 (1962). It has been reported that fewbacterial species have been subjected to more intensive investigationduring the century than Streptococcus pyogenes or the group Astreptococcus. As conviction grew that this organism was first theprincipal, and then the exclusive agent for acute rheumatic fever,researchers sought with much determination and effort to dissect out theproduct of the bacterium whose toxic or antigenic components might touchoff the rheumatic process. For a thorough study of rheumatic fever andstreptococcal infection See Stollerman, Rheumatic Fever andStreptococcal Infection (New York Clinical Cardiological Monographs,Grune and Stratton, 1975). The central role of M protein in immunityagainst group A streptococci has been reviewed by Stollerman. Referenceis also made to Beachey and Seyer, "Primary Structure andImmunochemistry of Group A Streptococcal M Proteins," Seminars inInfectious Disease, Vol.4, pp. 401-410 (J. B. Robbins, J. C. Hill and J.C. Sadoff, eds., Georg Thiemeverlag, pub., New York and Stuttgart,1982).

Most efforts to develop a vaccine were frustrated by severe toxicreactions to almost any Streptococcal product introduced into the humanhost. Some of these products have been shown to give rise to antibodiesthat cross react with host tissues, especially the heart. Kaplan andMeyerserian, "An Immunological Cross-Reaction Between Group AStreptococcal Cells and Human Heart Tissue," Lancet, Vol. i, pp. 706-710(1962); Zabriskie and Freimer, "An Immunological Relationship Betweenthe Group A Streptococcus and Mammalian Muscle," J. Exp. Med, Vol. 124,pp. 661-678 (1966). Although it has been long established that the Mprotein on the surface of group A Streptococcus contains the protectiveantigens of these organisms, the fear has been that the isolated Mprotein may be associated with potentially harmful tissue cross-reactiveantigens that give rise to rather than prevent, rheumatic fever. Thisfear has been perpetuated by the finding that certain rheumatogenicStreptococci produce M proteins that are closely associated with a heartcross-reactive antigen. Kaplan, "Immunologic Relation of Streptococcaland Tissue Antigen. I. Properties of an Antigen in Certain Strains ofGroup A Streptococci Exhibiting an Immunologic Cross-Reaction with HumanHeart Tissue," J. Immunol. Vol. 90, p. 595 (1963). Indeed, recently ithas been established that one of the M protein molecules contains,within its covalent structure, an epitope that elicits a protectiveanti-Streptococcal antibody that also cross-reacts with a sarcolemmalprotein of human heart tissue. Dale and Beachey, "Protective AntigenicDeterminant of Streptococcal M Protein Shared with Sarcolemmal MembraneProtein of Human Heart," J. Exp. Med., Vol. 156, pp. 1165-1176 (1982).

U.S. Pat. No. 4,284,537, to E. Beachey, issued Aug. 18, 1981, disclosedthe amino acid sequence of two peptide fragments derived from type 24 Mprotein. It also disclosed that each of these natural fragments, whencovalently linked to a carrier such as a polylysine, was able to elicittype-specific opsonic antibodies effective against Streptococcuspyogenes. Each of these fragments was a natural extract, and eachcontained 35 amino acids.

U.S. Pat. No. 4,454,121, to E. Beachey, issued Jun. 12, 1984, discloseda synthetic peptide (S-CB7) and that one of the protective determinantsis located in a specific fragment of S-CB7 of type 24 M protein whichcontains only twelve amino acid residues (S-CB7(18-29)). S-CB7, asdescribed, differs from the native CB-7 fragment in that theCOOH-terminal residue of S-CB7 is methionine, in contrast to homoserine.The specification also teaches and described covalently linkedconjugates of S-CB7 and appropriate hapten carriers, natural, like BSAor OVA or synthetic, like polylysine. Further details about this workhave been published in Nature on Jul. 30, 1981, by Beachey et al, 292,pages 457-459.

U.S. Pat. No. 4,521,334, entitled "Synthetic Polypeptide Fragments," toEdwin H. Beachey, issued Jun. 4, 1985, discloses the amino acid sequenceof three peptide fragments CB3, CB4, and CB5, and 35 and 37 amino acidsequences of type 24 M which contain antigenic determinantscorresponding to the antigenic determinants contained in CB3-CB7. U.S.Pat. No. 4,597,967 entitled "Synthetic Polypeptide Fragments," to EdwinH. Beachey, issued Jul. 1, 1986, discloses that these fragments, whencovalently linked to a carrier such as polylysine, are able to elicittype-specific opsonic antibodies effective against Streptococcuspyogenes.

U.S. application Ser. No. 739,963 entitled "Biologically Active HybridPeptides of Streptococcal M Protein and Compositions and use" to EdwinH. Beachey et al filed May 31, 1985 disclosed peptide sequencescontaining fragments of M5, M6, and M24 proteins which are able toelicit opsonic and bactericidal antibodies to Streptococcus pyrogeneswhich are not serologically cross-reactive with tissue antigens of thehuman or host heart. U.S. application Ser. No. 839,750 entitled"Synthetic M Proteins-Streptococci Type 6" to Edwin H. Beachey, et alfiled Mar. 14, 1986 discloses the synthesis of type M6 protein antigenconjugates. U.S. application Ser. No. 858,436 entitled "Localization ofProtective Epitopes of the Amino Terminus of Type S Streptococcal MProtein," to Edwin H. Beachey, et al filed May 1, 1986 disclosed thesynthesis of Type M5 protein antigen conjugates.

The above patents disclose small peptide fragments which are immunogenicand contribute to the development of a safe and effective vaccineagainst those streptococcal infections that initiate fevers andrheumatic heart disease. The approach was that very small peptides wouldpermit disposal of a large portion of the M protein molecule andtherefore, should reduce the chances of eliciting immunologicalcross-reactions against host tissues. See for instance theabove-referred to U.S. Pat. No. 4,454,121 Columns 1 and 2.

For additional information regarding type specific protective immunityevoked by synthetic peptides of Streptococcus pyogenes M protein, See,Beachey, et al., "Type Specific Protective Immunity Evoked by SyntheticPeptide of Streptococcus pyogenes M Protein," Nature, Vol. 292, No.5822, pp 457-459 (Jul. 30, 1981).

For additional literature in this field See Hasty, et als, "HybridomasAntibodies Against Protective and Non-Protective Antigenic Determinantsof a Structurally Defined Polypeptide Fragment of Streptococcal MProtein," J. Exp. Med. Vol. 155, p 1010 (April 1982); and Hopp andWoods, "Prediction of Protein Antigenic Determinants from Amino AcidSequences," Proc. Natl. Acad. Aci. USA, Vol. 78, No. 6, pp. 3824-3828(June 1981).

Notwithstanding these advances, there remains a serious need, as yetunfilled for an orally administrable vaccine incorporating thesenon-cross-reactive immunogenic polypeptides. By administering thesepeptides in the form of an attenuated non-virulent recombinant bacteriumcapable of their synthesis, the present invention marks another forwardstep and provides another advance in the medical sciences, particularlyin the control of Streptococcal infections.

Numerous serotypes of M proteins are known, coded on genes that arealleles of each other. Each serotype corresponds to a different strainof S. pyogenes, and these serotypes differ only by their amino terminalsequences.

The present invention relates in a general way to a genetic engineeringapproach to the synthesis of a Streptococcal M protein antigen orantigen fragment which is effective to elicit opsonic antibodies againstStreptococcal infections.

In a more specific sense the invention provides a transformednon-virulent bacterium which contains a heterologous nucleotide sequencewhich nucleotide sequence is encoded for and expresses a Streptococcal Mprotein antigen.

The invention also provides a transformed non-virulent host in which theexpressed Streptococcal M protein antigen is effective to elicit opsonicantibodies against streptococcal infection.

Another object of the invention is to also provide a protein antigenwhich is effective against virulent forms of the non-virulent bacterium.

A specific object of the invention is to provide a transformednon-virulent bacteria of the genus Salmonella, specifically thetyphimurium species, which carries a plasmid which encodes an M proteingene, more specifically the type 5 M protein gene.

Another object of the invention is to provide a Streptococcal M proteinantigen which is effective to elicit opsonic antibodies that conferspecific immunity against streptococcal infections.

An object of this invention is to provide a multivalent Streptococcal Mprotein antigen which is effective to elicit opsonic antibodies againstmore than one serotype M Streptococcal strain.

Another object of the invention is to provide an expressed StreptococcalM protein antigen which is expressed in the cytoplasm and not expressedon the surface of the bacterium.

It is a further object of the invention to provide a Streptococcal Mprotein antigen which when expressed by the host, particularly anon-virulent heterologous microbial host into which the gene for theStreptococcal M protein antigen has been cloned, is an effective antigenwhich elicits immunity and is not serologically cross-reactive withhuman tissue antigens, especially those for the heart. A further objectof the invention is a transformed non-virulent bacterium which is of thenon-group A species of the genus Streptococcus.

Another object of this invention is a transformed host, e.g., abacterium, particularly a non-virulent enteric bacterium, which, despiteits non-virulence, will still elicit antibodies. It is an object of thisinvention to provide a transformed non-virulent host bacterium that willnot colonize in the subject to be immunized, but will multiply to thelimited extent necessary to elicit the antibodies.

An object of this invention is the transformation of Salmonellatyphimurium. Another object of this invention is the transformation ofStreptococcus sanguis.

Another object of the invention is a transformed non-virulent hostbacterium into which a heterologous nucleotide sequence of anotherbacterial species has been cloned, which nucleotide sequence is encodedfor and expresses the protein antigen which is effective to elicitopsonic antibodies against the other bacterial species.

Another important object of the invention is to provide the nucleotidesequences carried by the gene which codes for and expresses the antigensof the other bacterial species.

Another object of the invention is to provide various other geneticallyengineered components leading to the transformed non-virulent host.

Another object of the invention is to clone into the host bacterium, thenucleotide sequences encoded for immunogenic polypeptides effective toelicit opsonic antibodies against Streptococcal infections, inparticular for Streptococcal serotypes 5, 6 and 24.

Another object of the invention is to clone into the host bacterium, thenucleotide sequences encoded for an E. coli surface antigen that elicitsanti-adhesive antibodies.

Another object of the invention is the genetic engineering steps whichleads to the preparation of the non-virulent bacteria.

Another object of the invention is the manufacture of plasmids andobject constructs necessary to clone into the S. typhimurium strain theheterologous nucleotide sequence that expresses the desiredStreptococcal M protein antigens. An important object of the inventionis the manufacture of plasmids that are stably expressed in Aro⁻ S.typhimurium SL3261.

Another object of the invention is a method of immunization of a mammalsuch as a human in which there is administered to that mammal is adosage effective to elicit opsonic antibodies and confer systemicimmunity against streptococcal infections, the transformed non-virulentbacterium described above containing the heterologous nucleotidesequence. Plasmids (and vectors) are also objects of the invention.

An important object of the invention is the oral administration of thecomposition of this invention, although other administrations are alsoprovided by this invention including by parental route.

An important aspect of the invention is to provide immunity against oralinfections of the streptococcal type.

Another aspect of the invention is to provide a non-virulent compositionwhich is very well tolerated by the subject to which the composition isadministered.

It is a noteworthy aspect of the invention that the immunity to thepatient is systemic and is conferred comparatively rapidly andcompletely even when administered orally.

Other objects of the invention will become apparent from the descriptionwhich follows. Other features and advantages of the invention willappear from the examples which follow and by referring to the appendeddrawing in which:

FIG. 1 shows Immunoblot analysis of type 5M protein expressed by pMK207-transformed Salmonella typhimurium LB5000 (Lane 1) and SL3261 (Lane2-9).

In addition to the patents and other publications mentioned above, otherprior art which has been taken into consideration in the description ofthis invention includes:

Beachey, et als., "Repeating Covalent Structure and ProtectiveImmunogenicity of Native and Synthetic Polypeptide Fragments of Type 24Streptococcal M Protein," J. Biol. Chem, Vol. 258, No. 21 pp13,250-13,257 (1983).

van de Rijn, et als., "Group A Streptococcal Antigens Cross-Reactivewith Myocardium," J. Exp. Med., Vol. 146, pp. 579-599 (1977).

van de Rijn, et als., "Immunochemical Analysis of Intact M ProteinSecretd From Cell Wall-Less Streptococci," Infect. Immun., Vol. 32, pp.86-91 (1981).

Edman and Begg, "A Protein Sequenator," European J. Biochem. Vol. 1, pp.80-91 (1967).

Phillips, et als., "Streptococcal M Protein: Helical Coiled - CoilStructure and Arrangement on the Cell Surface," Proc. Natl. Acad. Sci.USA, Vol. 78, No. 8 pp. 4689-4693 (August 1981).

Laver, et als., ∂Antigenic Drift in Type A Influenza Virus: PeptideMapping and Antigenic Analysis of A/PR/8/34(HON1) Variants Selected WithMonoclonal Antibodies," Proc. Natl. Acad. Sci. USA, Vol. 76, No. 3, pp.1425-1429 (March 1979).

Atassi, "Antigenic Structure of Myoglobin: The Complete ImmunochemicalAnatomy of a Protein and Conclusions Relating to Antigenic Structures ofProteins," Immunochemistry, Vol. 12, pp. 423-438 (1975).

Kabat, Structural Concepts in Immunology and Immunochemistry, pp. 89-100(Holt, Rhinehart & Winston, New York, (1968).

Nisonoff, Methods in Immunology and Immunochemistry, Vol. 1, pp. 120-187(1977).

Munoz, Methods in Immunology and Immunochemistry, Vol. 3, pp. 146-160(1970).

Manjula and Fischetti, "Tropomyosin-like Seven Residue Periodicity inThree Immunologically Distinct Streptococcal M Proteins and itsImplications for the Antiphagocytic Property of the Molecule," J. Exp.Med., Vol. 155, pp. 695-708 (1980).

Beachey and Stollerman, "Toxic Effects of Streptococcal M Protein onPlatelets and Polymorphonuclear Leukocytes in Human Blood," J. Exp.Med., Vol. 134, pp. 351-365 (1971).

Dale, et als., "Heterogenecity of Type-Specific and Cross-ReactiveAntigenic Determinants Within a Single M Protein of Group AStreptococci," J. Exp. Med., Vol. 155, pp. 1026-1038 (1980).

Beachey, et als. "Purification and Properties of M Protein Extractedfrom Group A Streptococci with Pepsin: Covalent Structure of the AminoTerminal Region of Type 24 M Antigen," J. Exp. Med. Vol. 145 pp.1469-1483 (1977).

Beachey, et als., "Primary Structure of Protective Antigens of Type 24Streptococcal M Protein," J. Biol. Chem., Vol. 255, pp. 6284-6289(1980).

Beachey, et als., "Repeating Covalent Structure of Streptococcal MProtein," Proc. Natl. Acad. Sci. USA, Vol. 75, pp. 3163-3167 (1978).

Beachey, et als., "Human Immune Response to Immunization with aStructurally Defined Polypeptide Fragment of Streptococcal M Protein,"J. Exp. Med., Vol. 150, pp. 862 (1979).

Brown, et al., "An Attenuated aroA Salmonella typhimurium VaccineElicits Humoral and Cellular Immunity to Cloned -Galactosidase in Mice,"The Journal of Infectious Diseases, Vol. 155, No. 1 (January 1987). Thispublication discusses Salmonella typhimurium strain SL3261, and anattenuated aroA vaccine strain, which was used as a carrier for theplasmid pXY411.

This publication of Brown, et al., is incorporated herein by reference.Another publication of interest is Hoiseth and Stocker,"Aromatic-dependent Salmonella typhimurium are Non-Virulent andEffective as Live Vaccines," Nature, Vol. 291 (May 21, 1981); Smith, etal., "Aromatic-Dependent Salmonella typhimurium are Non-Virulent andEffective as Live Vaccines," Am. J. Vet. Res, Vol. 45, No. 1 (January1984); Smith et al., "Vaccination of Calves Against Salmonella Dublinwith Aromatic-Dependent Salmonella typhimurium," Am. J. Vet. Res., Vol.15, No. 11 (November 1984); Maskell, et al., "Attenuated Salmonellatyphimurium as Live Oral Vaccines and Carriers for Delivering Antigensto the Secretory Immune System," Vaccines 86 (Cold Spring HaborLaboratory 1986).

Patents in the genetic engineering field which are of general interestinclude:

U.S. Pat. No. 4,428,941 entitled "Nucleotide Sequence Coding the Surfaceof the Hepatitis B Virus, Vector Containing Said Nucleotide Sequence,Process Allowing the Obtention Thereof and Antigen Obtained Thereby,"issued to Francis Galibert, et als., on Jan. 31, 1984; U.S. Pat. No.4,518,584 entitled "Human Recombinant Interleukin-2 Muteins," issued toMark, et als., on May 21, 1985; U.S. Pat. No. 4,588,585 entitled "HumanRecombinant Cysteine Depleted Interferon-B Muteins," issued to Mark etals., on May 13, 1986; and U.S. Pat. No. 4,625,252 entitled "RecombinantBacterial Plasmids Containing the Coding Sequences of Insulin Genes,"issued to Rutter, et als., on Mar. 24, 1987.

U.S. Pat. No. 4,666,846 entitled "Novel Cloning Vectors ContainingSelectable Genetic Markers for Use in Streptomyces and RelatedOrganisms," issued to Fayerman, et als. on May 19, 1987; and U.S. Pat.No. 4,666,847 entitled "Recombinant DNA Means and Methods," issued toAlford, et als., on May 19, 1987.

In accordance with the invention it is preferable that the plasmid whichencodes the M protein gene be cloned first and expressed in Escherichiacoli. Any other enteric bacilli of the coliform group such as Klebsiellaor Enterobacter can be used, but normally E. coli is preferred.Therefore the plasmid carrying the M gene is isolated and purified andthen a construct is built to transform the desired non-virulentbacteria, such as the aroA-S. typhimurium (SL3261). It is to be notedthat this mutant strain exhibits a nutritional marker both for PABA and2,3-DHB. See Brown et al., cited above. It is to be noted that anotherdesired specie of S. typhimurium is recA⁻ S. typhimurium, particularlystrain Ty21a. See Clements, et al., "Construction of a Potential LiveAro Vaccine for typhoid type fever and cholorea--E. coli--relateddiarrheas," Infect. Immun., 46:564-9 (1984). Also see the otherreferences cited in the above cited Brown, et al., article, which arealso incorporated herein by reference.

It is preferred to obtain the M protein gene from a virulent strain ofS. pyogenes. However, it is possible to obtain the gene from anattenuated, non-virulent strain of S. pyogenes, or even to fabricate thenucleotide sequence coded for the desired M protein.

The recombinant DNA cloning vectors of the present invention are notlimited for use in a single species or strain of Salmonella. To thecontrary, the vectors are broadly applicable and can be transformed inhost cells of other gram negative bacteria such as of theEnterobacteriaceae genus (such as Shigella and Klebsiella like(Klebsiella penumoniae, Enterobacter like Enterobacter aerogenes).Salmonellae, such as Salmonella arizona, and Citrobacter may be used ifappropriately rendered non-virulent or attenuated.

Common Salmonella species which may be used when attenuated and renderednon-virulent include the following: S. paratyphi A, S. schottmulleri, S.typhimurium, S. paratyphi C, S. choleraesuis, S. montevideo, S. newport,S. typhi, S. enteritidis, S. gallinarum, S. anatum.

In accordance with the invention there may also be used as host for therecombinant DNA cloning vectors of the present invention bacteria of theStreptococcus genus which are non-virulent or which have been madenon-virulent or attenuated, including Streptococci of the immunologicalgroups A-O but generally other than A. Suitable Streptococci which canbe used as bacterial host include S. cremoris, S. faecalis, S.salivarius, S. mitior, S. mitis, S. mutans and S. sanguis, the latterspecies is presently a preferred specie.

Additional appropriate microorganisms which may be attenuated andtransformed in accordance with the invention are known. Reference may bemade to Davis, et al., Microbiology, (Harper & Row, Second edition,1973).

Generally any enteric bacterium may serve as the host bacterium. It ispreferable that the host bacterium only survive in the subject longenough to elicit the opsonic response, but generally any bacterialstrain that has been attenuated not to colonize but will still multiplyto a limited degree to elicit antibodies to the foreign protein can beused. In a preferred embodiment of the invention the Aro⁻ strain of S.typhimurium is used, which requires two metabolites not found inmammalian tissues, PABA and 2,3-DHB. As a result, the innoculatedbacteria die after several generations from lack of these metabolites.See Hoiseth and Stocker, cited above.

However, any mutated microbial agent with a metabolic deficiency fornutritional compounds not found in the tissues of the subject to beimmunized, or one so made by genetic manipulations, may be employed.

It is to be noted that the non-virulent aro⁻ Salmonella typhimuriumSL3261 into which a plasmid containing the structural gene encoding theserotype 5M protein antigen has been transformed expresses the entire M5 protein molecule, which expression is confined almost exclusively tothe S. typhimurium cytoplasmic compartment. It is a unique andunexpected aspect of this invention that an immunogenic and protectivesurface antigen such as the Streptococcal M protein antigen is expressedin the cytoplasm of the non-virulent host bacterium.

Thus it can be seen that in accordance with the invention the desirednucleotide sequence which codes for and expresses the protein antigenwhich is effective to elicit opsonic antibodies against streptococcalinfections particularly of the serotype 5, can be cloned into a varietyof hosts. In a broader sense therefore the transformed host in which thenucleotide sequence is found after replication need not be heterologouswith respect to the nucleotide sequence, nor does the sequence need tobe heterologous with respect to the microorganisms.

In accordance with a specific embodiment of the method of immunizationof a warm-blooded animal, it has been showed that a) peroraladministration of up to 1.65×10⁹ mutant non-virulent Salmonellacontaining the plasmid pMK207 encoding serotype 5 Streptococcal Mprotein was well tolerated in mice; b) plasmid mPK207 was extremelystable both in vitro and in vivo; c) the mice receiving the highest dose(10⁹) of bacteria harbored the microorganisms in the liver for as longas three weeks without ill effects; d) the mice immunized orally withnon-virulent transformed Salmonella expressing the serotype 5Streptococcal M protein antigen gene developed opsonic serum antibodiesas early as three weeks against serotype M5 Streptococci; and e) theimmunized mice were completely protected at three weeks againstintra-peritoneal challenges of the homologous serotype M5 (but not theheterologous serotype M24) Streptococci. It is noteworthy that nocross-reactive immunity is observed when the composition of theinvention is administered orally. The cytoplasmic expression of the Mprotein antigen in the non-virulent bacterium is especially advantageousfor this oral administration. The antigen is protected within thecytoplasm of the non-virulent bacterium from the acids of the stomachand other damaging agents until the non-virulent cell dies and releasesthe antigen, ordinarily in the small intestine, the preferred locationfor delivery of the antigens.

In accordance with the invention the non-virulent bacterium may also beused as a host for recombinant DNA cloning vectors containing nucleotidesequences which code for and express immunogenic polypeptides which arespecifically effective to confer immunity against Streptococcalinfections and which are not cross-reactive with human tissue antigens,especially those of the heart.

Immunogenic polypeptides in accordance with the invention includesynthetic oligopeptides copying regions of M protein molecules lackingauto-immune epitopes, as described in U.S. Pat. No. 4,284,537 issued toE. Beachey on Aug. 18, 1981, U.S. Pat. No. 4,454,121 issued to E.Beachey on Jun. 12, 1984, U.S. Pat. No. 4,521,334 issued to E. Beacheyon Jun. 4, 1985, U.S. Pat. No. 4,597,967 issued to E. Beachey on Jul. 1,1986, U.S. application Ser. No. 739,963, on E. Beachey, et al, filed May31, 1985, U.S. application Ser. No. 839,750 to E. Beachey et al filedMar. 14, 1986 and U.S. application Ser. No. 858,436 to E. Beachey, et alfiled May 1, 1986, all cited above and incorporated by reference.

The capacity of serotype M 24 polypeptides as a carrier for a highlymulti-valent vaccine is being tested with other serotype M polypeptidefragments.

Another approach of the invention has been to use Streptococcus sanguisbacteria which have been transformed with the M protein - expressingplasmids. The expression of serotype type 5 M protein in S. sanguis hasbeen accomplished. The M protein gene was carried on a shuttle vectorplasmid and transformed into the host bacterium, which was shown toexpress serotype 5 M protein fibrils on the surface of the organism.Moreover, the organism were able to bind fibrinogen which in turnrendered the microorganism resistant to phagocytosis.

In accordance with the invention a desirable method for immunizingagainst Streptococcal infections is to instill attenuated S. sanguis,intranasally to evoke local immune responses at the very site whereStreptococci commonly enter the host.

Another approach of the invention has been to clone into the hostorganism, together with the M protein - expressing plasmid, a plasmidexpressing the antigen of a third bacterial species. In particular, apSH-2 plasmid cloned from the CSH50 strain of E. coli has been clonedinto Sal. typhimurium causing the host to express an E. coli 29kilodalton surface antigen that elicits Fim H antiadhesive antibodies.These antiadhesive antibodies are not only effective against E. coli,they are also effective against other gram negative bacteria.

Other advantages characteristic of the invention will appear from thenon-limiting examples which follow.

EXAMPLE 1 TRANSFORMATION OF NON-VIRULENT HOST BACTERIUM TO EXPRESSSTREPTOCOCCAL M PROTEIN

The aro⁻ strain of Salmonella typhimurium SL3261, developed by Stockerand Hoseith (cited above) was chosen because this mutant form ofmicrobial agent is capable of invasion but not causing disease. Thismutant strain exhibits a nutritional marker for both p-aminobenzoic acid(PABA) and 2,3-dihydrobenzoic acid (DHB). See Stocker and Hoseith, citedabove.

The M5 protein antigen structural gene (smp 5) was cloned and expressedin E. coli LE392 as described by Kehoe, et als., "Cloning and GeneticAnalysis of Serotype 5M Protein Determinant of Group A Streptococci:Evidence for Multiple Copies of the M5 Determinant in the Streptococcuspyogenes Genome," Infect Immun., Vol. 48, pp 190-197 (1985) and Poirer,et als., "Expression of Protective and Cardiac Tissue Cross-ReactiveEpitopes of Type 5 Streptococcal M Protein in Escherichia coli," Infect.Immun. Vol. 48, pp. 198-203 (1985), both of which are incorporatedherein by reference.

The plasmid pMK207 was isolated, purified, and transformed first into anrm+ strain LB5000 of Sal. typhimurium, as described by Bullas nd Ryo,"Salmonella typhimurium LT2 Strains which are r⁻ m+ for all ThreeChromosomally Located Systems of DNA Restriction and Modification," J.Bacteriol, Vol. 156, pp. 471-474 (1983) and Lederberg and Cohen,"Transformation of Salmonella typhimurium by Plasmid DeoxyribonucleicAcid," J. Bacteriol., Vol. 119, pp. 1072-1074 (1974), both of which areincorporated herein by reference. The plasmid isolated and purified fromLB5000 was then used to transform the aro⁻ Sal. typimurium SL3261 inusing the same above-cited procedures described by Lederberg and Cohenand Bullas and Ryo.

The transformed LB5000 and SL3261 expressed the entire M5 proteinmolecule as demonstrated by Western blot analysis of whole cell lysates(FIG. 1; lanes 1 and 2). The typical triplet of M5 protein (See Poirer,et als., and Kehoe, et als, cited above) migrated as bands of M_(r) 59,56 and 54k. Preliminary studies designed to determine the location ofthe M5 protein as expressed by Sal. typhimurium SL3261 indicated thatthe recombinant protein was confined almost exclusively to thecytoplasmic compartment. The M5 protein was expressed in a stablefashion by SL3261 even in the absence of antibiotic pressure, i.e.,expression was apparent following repeated subscultures over five days,which represents approximately 35 generations of growth (FIG. 1; lanes3-8).

EXAMPLE 2 MOUSE TOLERANCE OF TRANSFORMED SAL. TYPHIMURIUM SL3261

In preliminary studies, BALB/c mice were challenged with increasingdoses of SL3261-pMK207 to determine whether or not the animals wouldtolerate the transformed organisms. None of the mice receiving up to amaximum of 1.65×10⁹ organisms per oral dose either sickened or died. At1, 3, 5 and 10 weeks after innoculation, two mice from each dosage groupwere sacrificed and their livers, spleens and intestines cultured forSL3261-pMK207 on McConkey's agar containing 50 mg/ml kanamycin sulfateand 10 mg/ml each of PABA and DHB. Non-lactose fermenting colonies couldbe isolated at one week only from the mice receiving 10⁶ organisms ormore, and at three weeks, only from those receiving 10⁹ organisms (Table1). No isolates were recovered after three weeks. The colonies isolatedat three weeks expressed an intact M5 protein, which suggests that smp5was stable in vivo (FIG. 1; lane 9). The 1, 3, 5, 9, 10, 15 and 20 weekssera from mice innoculated orally were shown to exhibit antibodiesagainst type 5M protein and type 5 Streptococci by ELSIA andopsonization experiments, respectively (Table 2). The saliva obtainedfrom mice that were 20 weeks post-immunization was also shown to possessanti-M5 protein antibodies, primarily of the IgA class (Table 3).

                                      TABLE 1                                     __________________________________________________________________________    DISTRIBUTION OF SALMONELLA TYPHIMURIUM SL3261 IN                              THE ORGANS OF BALB/c MICE AFTER ORAL IMMUNIZATION                             INOCULATION                                                                             SALMONELLA DISTRIBUTION IN ORGANS.sup.2 :                           DOSE.sup.1                                                                              INTESTINE      LIVER - GALL BLADDER                                                                         SPLEEN                                Initial                                                                            Boost                                                                              Week 1                                                                             Week 2                                                                             Week 3                                                                             Week 1                                                                             Week 2                                                                             Week 3                                                                             Week 1                                                                             Week 2                                                                             Week 3                      __________________________________________________________________________    1.7 × 10.sup.9                                                               1.1 × 10.sup.9                                                               2.1 × 10.sup.4                                                               <50  <50  4 × 10.sup.2                                                                 3 × 10.sup.2                                                                 <50  4 × 10.sup.2                                                                 <50  <50                         1.7 × 10.sup.8                                                               1.1 × 10.sup.8                                                               <200 <50  .sup. ND.sup.3                                                                     8 × 10.sup.2                                                                 <50  ND   4 × 10.sup.2                                                                 <50  ND                          1.7 × 10.sup.7                                                               1.1 × 10.sup.7                                                               <200 <50  ND   1 × 10.sup.2                                                                 <50  ND   1 × 10.sup.2                                                                 <50  ND                          1.7 × 10.sup. 6                                                              1.1 × 10.sup.6                                                               <100 ND   ND   <50  ND   ND   <50  ND   ND                          1.7 × 10.sup.5                                                               1.1 × 10.sup.5                                                               <100 ND   ND   <50  ND   ND   <50  ND   ND                          1.7 × 10.sup.4                                                               1.1 × 10.sup.4                                                               <100 ND   ND   <50  ND   ND   <50  ND   ND                          __________________________________________________________________________     .sup.1 Mice were given serially 10fold diluted oral doses of S.               typhimurium strain SL3261 beginning with 1.7 × 10.sup.9 colony          forming units (cfu) on day 1 (initial dose), with an oral booster dose        beginning with 1.1 × 10.sup.9 cfu on day 5. Each dose was suspended     in 25 μl of phosphate buffered saline (PBS, pH 7.2) containing 5 μg     ml.sup.-1 kanamycin sulfate, and 1 μg ml.sup.-1 each of paraminobenzoi     acid (PABA) and 2,3dihydroxybenzoic acid (DHB). Mice were sacrificed at 1     2, and 3 weeks after administration of the initial dose.                      .sup.2 Intestine (pyloric sphincter to rectum), livergall bladder, and        spleen were removed aseptically, placed in PBS, homogenized with a            Cellector ™ (100 mesh; Bellco), and plated onto MacConkey agar             containing 10 μg ml.sup.-1 each of PABA and DHB with or without 50         μg ml.sup.-1 of kanamycin.                                                 .sup.3 Not determined.                                                   

                  TABLE 2                                                         ______________________________________                                        Opsonization of M Type 5 S. pyogenes with Mouse                               Anti-M5-Protein-Serum Obtained from BALB/c Mice                               Orally Immunized with S. typhimurium                                          SL3261-pMK207 and their Anti-pepM Protein ELISA Titers.                                             Percent                                                           ELISA Titer.sup.1                                                                         Opsonization.sup.2                                      Test Serum  IgA    IgG     IgM  Type 5                                                                              Type 24                                 ______________________________________                                        Pre-Immune  <50    <50     <50   0    2                                        1 week     100    200     <50   4    4                                        3 weeks    400    400     <50  36    0                                        5 weeks    800    800     <50  52    2                                        9 weeks    100    1600    200  90    2                                       10 weeks    50     800     400  92    2                                       15 weeks    100    800     400  52    0                                       15 weeks.sup.3                                                                            800    12800   800  82    0                                       20 weeks    <50    400     200  10    2                                       20 weeks.sup.3                                                                            400    3200    200  80    2                                       Anti-pepM5.sup.4                                                                          ND.sup.5                                                                             ND      ND   96    2                                       Anti-pepM24.sup.4                                                                         ND.sup.                                                                              ND      ND    0    98                                      ______________________________________                                         .sup.1 ELISA titers were determined using immobilized pepM5 reacted with      BALB/c mouse antiSL3261-pMK207-sera followed with peroxidase conjugated       goat antimouse IgA, IgG, or IgM. All mouse antisera reacted against           immobilized pepM24 gave titers of <50.                                        .sup.2 Opsonization was determined as the percent of total neutrophils        with associated streptococci.                                                 .sup.3 Mice were boosted 48 hours prior to bleed out with 50 μg of         pepM5 in 0.1 ml of phosphate buffered saline, pH 7.2.                         .sup.4 Both antipepM5 and antipepM24 were prepared in rabbits to serve as     positive (homologous reaction) and negative (heterologous reaction)           controls.                                                                     .sup.5 The antibody titers for the rabbit antipepM5 or antipepM24 were no     determined.                                                              

                  TABLE 3                                                         ______________________________________                                        Salivary Antibody Response.sup.1 Against M5-Protein in BALB/c                 Mice Immunized Orally with S. typhimurium SL3261-pMK207.                                    ELISA Titer.sup.2 Against pepM5                                 Test Serum      IgA    IgA + IgG + IgM                                        ______________________________________                                        Pre-Immune      <4     <4                                                     20 Weeks.sup.3  32      32                                                    20 Weeks + Boost.sup.4                                                                        64     128                                                    ______________________________________                                         .sup.1 Saliva was collected and peeled from mice (four per group)             following induction intraperitoneally with 0.1 ml of 2% pilocarpine.          .sup.2 ELISA titers were determined using immobilized pepM5 reacted with      saliva (serially diluted twofold) collected from BALB/c mice immunized        with SL3261pMK207 followed with peroxidase conjugated goat antimouse IgA      or antimouse IgA + IgG + IgM mixture.                                         .sup.3 Mice were immunized 20 weeks prior to the saliva collection (see       Table 2).                                                                     .sup.4 Mice were injected with a booster dose of 50 μg of pepM5 in 0.1     ml of phosphate buffered saline, pH 7.2, 60 hours prior to saliva             collection.                                                              

EXAMPLE 3

A group of mice was innoculated orally with two doses of SL3261-pMK207and challenged 22 days after the first dose with type 5 or 24Streptococci; or the virulent parent Sal. typhimurium 1344 (Table 3). Ascan readily be seen by the results, the mice receiving the M5 expressingSal. typhimurium mutant were completely protected against anintra-peritoneal challenge of type 5 Streptococci, but not with type 24Streptococci. Control mice challenged intra-peritoneally with thevirulent SL1344 Sal. typhimurium were also protected. The type 5challenged mice were protected against a dose that exceeded the LD₅₀ by100-fold (Table 4). This protection against parenteral challenge by type5 Streptococci indicates that the immunity conferred is systemic.

                  TABLE 4                                                         ______________________________________                                        Challenge by Intra-Peritoneal Injection of                                    M Type 5 and Type 24 Streptococci or                                          S. typhimurium SL1344 in BALB/c Mice                                          Immunized Orally.sup.1 with Live aro.sup.-  S. typhimurium                    Transformed with pMK207 Expressing Type 5 M Protein.                          Challenge          Survival.sup.4,5                                           Organisms.sup.2                                                                            Dose.sup.3                                                                              Unimmunized                                                                              Immunized                                   ______________________________________                                        M5 Streptococci                                                                            1.7 × 10.sup.4                                                                    2/4        4/5                                         (Smith strain)                                                                             1.7 × 10.sup.5                                                                    0/4        5/5                                                      1.7 × 10.sup.6                                                                    0/4        5/5                                         M24 Streptococci                                                                           1.6 × 10.sup.3                                                                    1/3        1/3                                         (Vaughn strain)                                                                            1.6 × 10.sup.4                                                                    0/3        0/3                                                      1.6 × 10.sup.5                                                                    0/3        0/3                                         S. typhimurium                                                                             7.3 × 10.sup.1                                                                    0/3        3/3                                         (Strain SL1344)                                                                            7.3 × 10.sup.2                                                                    0/3        3/3                                                      7.3 × 10.sup.3                                                                    0/3        3/3                                         ______________________________________                                         .sup.1 Each immunized mouse received 1.2 × 10.sup.9                     pMK207transformed SL3261 S. typhimurium colony forming units (cfu) at 22      days and 1.6 × 10.sup.9 cfu at 17 days before challenged. Each dose     was administered orally suspended in 25 μl of phosphate buffered salin     containing 5 μg ml.sup.-1 kanamycin sulfate, and 1 μg ml.sup.-1 eac     of paraminobenzoic acid and 2,3dihydroxybenzoic acid.                         .sup.2 The LD.sub.50 for M5 and M24 streptocci, and SL1344 in BALB/c mice     was approximately 2 × 10.sup.4, 3 × 10.sup.3, and 1 ×       10.sup.0 CFU, respectively.                                                   .sup.3 Dose was determined as the CFU administered.                           .sup.4 Survival was recorded as the number of surviving mice divided by       the number of mice challenged.                                                .sup.5 All recorded deaths occurred between 3 and 6 days after challenge.     All surviving mice appeared healthy up to 30 days after challenge.       

A group of BALB/c mice which had been immunized orally withSL3261-pMK207 were challenged intra-nasally with type 5 or 24Streptococci, or the virulent parent Sal. typhimurium SL1344. As can beseen in Table 5, only those mice immunized orally with SL3261-pML207were able to survive an intra-nasal challenge with a dosage ofhomologous organisms which was otherwise sufficient to kill allimmunized animals. Further, the protection conferred by Sal. typhimuriumSL3261 expressing recombinant M5 protein in M type specific asdemonstrated by the inability of intra-nasally challenged mice totolerate M type 24 Streptococci. The fact that mice were refractory tochallenge by intra-nasal innoculation suggested that oral immunizationwith M5 protein expressing Sal. typhimurium SL3261 was sufficient toinpart local immunity.

                  TABLE 5                                                         ______________________________________                                        Challenge by Intra-Nasal Inoculation of M Type 5 and                          Type 24 Streptococci or S. typhimurium SL1344 in BALB/c                       Mice Immunized Orally.sup.1 with Live aro.sup.- S. typhimurium                Transformed with pMK207 Expressing Type 5 M Protein.                          Challenge          Survival.sup.4,5                                           Organisms.sup.2                                                                            Dose.sup.3                                                                              Unimmunized                                                                              Immunized                                   ______________________________________                                        M5 Streptococci                                                                            3.9 × 10.sup.7                                                                    0/4        6/6                                         (Smith strain)                                                                M24 Streptococci                                                                           3.5 × 10.sup.7                                                                    0/4        0/6                                         (Vaughn strain)                                                               S. typhimurium                                                                             4.3 × 10.sup.4                                                                    0/4        6/6                                         (Strain SL1344)                                                               ______________________________________                                         .sup.1 Each immunized mouse received 1.2 × 10.sup.9 pMK207              transformed SL3261 S. typhimurium colony forming units (cfu) at day 1 and     1.6 × 10.sup.9 cfu at day 5. Each dose was administered orally          suspended in 25 μl of phosphate buffered saline (PBS, pH 7.2)              containing 5 μg ml.sup.-1 kanamycin sulfate, and 1 μg ml.sup.-1 eac     of paraminobenzoic acid and 2,3dihydroxybenzoic acid.                         .sup.2 Challenge dose was determined as the cfu administered intranasally     in 20 μl of PBS (10 μl per nostril).                                    .sup.3 Mice were challenged 13 weeks after the initial immunization (ie.      day 1).                                                                       .sup.4 Survival was recorded as the number of surviving mice over the         number of mice challenged.                                                    .sup.5 Deaths from streptococci occurred between 2 and 4 days, while          deaths due to salmonella occurred between 5 and 7 days after challenge.       All surviving mice appeared healthy up to 30 days after challenge.       

The above examples are not to be construed as limitations, but aremerely illustrative of the invention. The invention also encompassescombinations of any number of immunogenic polypeptide sequences of anyStreptococcal serotype M protein antigen in a covalent linkage to anatural or synthetic carrier to create a multivalent vaccine broadlyprotective against serotype M Streptococcus.

The invention also encompasses utilizing an M protein polypeptidesequence as the carrier to which the other sequences are covalentlylinked, evoking an opsonic response to each one of the Streptococcalserotypes, the polypeptides of which are linked to the carrier, as wellas the Streptococcal serotype, the polypeptide of which is serving asthe carrier.

The invention also encompasses encoding a nucleotide sequence for such amultivalent polypeptide onto an S. pyogenes chromosome in the region ofthe M protein structureal gene, cloning it into plasmids andtransforming the plasmids into non-virulent host bacterium. Syntheticnucleotide sequences are preferred, but nucleotide sequences obtainedfrom living bacterial cells may also be employed. The invention alsoencompasses cloning into non-virulent host bacterium already transformedto express immunogenic polypeptides eliciting opsonic responses to typeM Streptococcal infections genetic material for the expression ofimmunogenic polypeptides eliciting opsonic responses to other virulentbacteria species and the covalent linkage of both species polypeptides.The invention also encompasses the resultant attenuated non-virulenthost organism, orally administrable as a broad-spectrum vaccine capableof eliciting opsonic responses to more than one species of bacteria.

One skilled in the art would have no difficulty in developing variousvariations in procedure and products which are within the spirit of theinvention and scope of the claims or their equivalent.

We claim:
 1. A method for immunizing a mammal against Streptococciinfections by eliciting opsonic antibodies to a Streptococcal M proteinantigen without eliciting antibodies which are cross-reactive with hearttissue antigens which comprises administering orally to said mammal inan amount effective to confer immunity against Group A Streptococciinfection, a therapeutic composition which comprises a biologicallyacceptable carrier and a non-virulent, live bacterium selected from thegroup consisting of Salmonella and Streptococcus sanguis, transformedwith a plasmid encoding the expressing a serotype-specific entireStreptococcal M protein immunizing antigen compartmentalizedintracellularly selected from the serotypes of the group consisting ofserotype 5, serotype 6 and serotype 24, releasing the antigen from thebacterium, whereby opsonic antibodies to the antigen of the sameserotype as the immunizing antigen elicited, without elicitingantibodies which are cross-reactive with heart tissue antigens, therebyconferring immunity against Streptococci infection to said mammal.
 2. Atherapeutic composition which comprises in an amount effective to elicitopsonic antibodies and to confer immunity against Group A Streptococciinfection in a mammal without eliciting antibodies which arecross-reactive with heart tissue antigens, a biologically acceptablecarrier and, a non-virulent, live Salmonella transformed with a plasmidencoding and expressing a serotype-specific entire Streptococcal Mprotein immunizing antigen compartmentalized intracellularly selectedfrom the group of serotypes 5, 6 and 24 which M protein is normallyserologically cross-reactive with human heart tissue antigens, whichcomposition upon oral administration to the mammal elicits opsonicantibodies to the antigen of the same serotype as the immunizing antigenas the antigen is released from the dying bacterium, thereby providingimmunity against Streptococci infection to said mammal.
 3. A therapeuticcomposition which comprises a biologically acceptable carrier and in anamount effective to elicit opsonic antibodies to a serotype-specificStreptococcal M protein selected from the group consisting of serotypes5, 6 and 24, a non-virulent, live transformed bacterium selected fromthe group consisting of Salmonella and Streptococcus sanguis whichcontains a plasmid encoding and expressing a serotype-specific entireStreptococcal M protein immunizing antigen compartmentalizedintracellularly, which composition upon oral administration to a mammalelicits opsonic antibodies to the antigen of the same serotype as theimmunizing antigen, as the antigen is released from the dying bacteriumwithout eliciting antibodies which are cross-reactive with human hearttissue antigens, thereby providing immunity to said mammal against GroupA Streptococci infection.
 4. A therapeutic composition which comprises abiologically acceptable carrier and in an amount effective to elicitopsonic antibodies to a serotype-specific Streptococcal M protein in amammal without eliciting antibodies which are cross-reactive with humanheart tissue antigens, a non-virulent, live transformed bacteriumselected from the group consisting of Salmonella and Streptococcussanguis expressing a serotype-specific entire Streptococcal M proteinimmunizing antigen compartmentalized intracellularly, which compositionupon oral administration to the mammal elicits opsonic antibodies to theantigen of the same serotype as the immunizing antigen, as the antigenis released from the dying bacterium, thereby providing immunity to saidmammal against Group A Streptococci infection.
 5. A method forimmunizing a mammal against Streptococci infection eliciting opsonicantibodies to a Streptococcal M protein antigen without elicitingantibodies which are cross-reactive with heart tissue antigens, whichcomprises administering orally to said mammal in an amount effective toconfer immunity against Streptococci infection, a therapeuticcomposition which comprises a biologically acceptable carrier and anon-virulent live, transformed bacterium selected from the groupconsisting of Salmonella and Streptococcus sanguis expressing aserotype-specific entire Streptococcal M protein immunizing antigencompartmentalized intracellularly, releasing the antigen from the dyingbacterium, whereby opsonic antibodies to the antigen of the sameserotype as the immunizing antigen are elicited, thereby providingimmunity to said mammal against Group A Streptococci infection.
 6. Thetherapeutic composition of claim 4 wherein the transformed bacterium isa plasmid-transformed bacterium.
 7. The therapeutic composition of claim6 wherein the bacterium is Streptococcus sanguis.
 8. The therapeuticcomposition of claim 6 wherein the bacterium has a mutational variationcausing a nutritional deficiency.
 9. The therapeutic composition ofclaim 8 wherein the bacterium is a Salmonella selected from the groupconsisting of the species S. paratyphi, S. schottmulleri, S.typhimurium, S. choleraesuis, S. montevideo, S. newport, S. typhi, S.enteritidis, S. gallinarum and S. anatum.
 10. The therapeuticcomposition of claim 9 wherein the Salmonella is typhymurium.
 11. Thetherapeutic composition of claim 6 wherein the immunity conferred issystemic.
 12. The therapeutic composition of claim 7 wherein theStreptococcus infectious challenge is mucosal.
 13. The therapeuticcomposition of claim 12 wherein the Streptococcus infectious challengeis intranasal.
 14. The method of claim 5 wherein the transformedbacterium is a plasmid-transformed bacterium.
 15. The method of claim 14wherein the bacterium is Streptococcus sanguis.
 16. The method of claim14 wherein the Salmonella is selected from the group consisting of thespecies S. paratyphi, S. schottmulleri, S. typhimurium, S. choleraesuis,S. montevideo, S. newport, S. typhi, S. enteritidis, S. gallinarum andS. anatum.
 17. The method of claim 16 wherein the Salmonella istyphymurium.
 18. The method of claim 14 wherein the immunity conferredis systemic.
 19. The method of claim 15 wherein the Streptococcusinfectious challenge is mucosal.
 20. The method of claim 19 wherein theStreptococcus infectious challenge is intranasal.
 21. The therapeuticcomposition of claim 3 wherein the bacterium is Streptococcus sanguis.22. The therapeutic composition of claim 3 wherein the bacterium is aSalmonella selected from the group consisting of the species S.paratyphi, S. schottmulleri, S. typhimurium, S. choleraesuis, S.montevideo, S. newport, S. typhi, S. enteritidis, S. gallinarum and S.anatum.
 23. The therapeutic composition of claim 3 wherein theSalmonella has a mutational variation causing a nutritional deficiency.24. The therapeutic composition of claim 23 wherein the bacterium isSalmonella typhimurium.
 25. The therapeutic composition of claim 24wherein the Salmonella is aro-Sal. typhimurium.
 26. The therapeuticcomposition of claim 25 wherein the aro-Sal. typhimurium is typhimuriumSL
 3261. 27. The therapeutic composition of claim 3 wherein theserotypes are selected from the group consisting of serotypes 5 and 6.28. The therapeutic composition of claim 27 wherein the serotype is 5.29. The therapeutic composition of claim 22 wherein the serotype is 6.30. The therapeutic composition of claim 3 wherein the serotype is 24.31. The therapeutic composition of claim 3 wherein the immunity issystemic.
 32. The therapeutic composition of claim 21 wherein theStreptococcus infectious challenge is mucosal.
 33. The therapeuticcomposition of claim 32 wherein the Streptococcus infectious challengeis intranasal.
 34. The therapeutic composition of claim 3 wherein the Mprotein is of serotype 5 and the Streptococcus infection of serotype 5.35. The therapeutic composition of claim 3 wherein the M protein is ofserotype 6 and the Streptococcus infection of serotype
 6. 36. Thetherapeutic composition of claim 3 wherein the M protein is of serotype24 and the Streptococcus infection of serotype
 24. 37. The method ofclaim 1 wherein the bacterium is Streptococcus sanguis.
 38. The methodof claim 1 wherein the Salmonella has a mutational variation causing anutritional deficiency.
 39. The method of claim 38 wherein theSalmonella is selected from the group consisting of the species S.paratyphi, S. schottmulleri, S. typhimurium, S. choleraesuis, S.montevideo, S. newport, S. typhi, S. enteritidis, S. gallinarum and S.anatum.
 40. The method of claim 39 wherein the bacterium is Salmonellatyphimurium.
 41. The method of claim 40 wherein the Salmonella isaro-Sal. typhimurium.
 42. The method of claim 41 wherein the Salmonellais aro-Sal. typhimurium is typhimurium SL
 3261. 43. The method of claim1 wherein the Salmonella multiplies in the mammal to a limited extentthen dies releasing the M protein antigen.
 44. The method of claim 1wherein the serotypes are selected from the group consisting ofserotypes 5 and
 6. 45. The method of claim 44 wherein the serotype isserotype
 5. 46. The method of claim 44 wherein the serotype is serotype6.
 47. The method of claim 1 wherein the serotype is serotype
 24. 48.The method of claim 1 wherein the immunity is systemic.
 49. The methodof claim 1 wherein the Streptococci infectious challenge is mucosal. 50.The method of claim 1 wherein the Streptococci infectious challenge isintranasal.